CuO nanoparticles for green synthesis of significant anti-Helicobacter pylori compounds with in silico studies

Helicobacter pylori (H. pylori) is a universal health intimidation as mentioned by the World Health Organization. The primary causal agent linked to a number of illnesses, including inflammation and the development of stomach ulcers, is Helicobacter pylori. Since, H. pylori develops antibiotic resistance quickly, current H. pylori treatment approaches are becoming less effective. Our research aims to highlight novel formulation antibiotics using CuO-NPs as catalysts and studied their activity as anti-helicobacter pylori supported by computational studies (POM analysis and molecular docking) software. They were designed for anti-Helicobacter Pylori action. All compounds revealed a bactericidal effect better than the reference McFarland standards.

heavily on heterogeneous catalysts 16 .It is expected that changing the support through ideologies like nanoscience and nanotechnology or keeping an eye on the pore structure would lead to the development of heterogeneous catalytic activity 17 .The issue of catalyst separation and recovery from the reaction matrix is addressed for heterogeneous catalysis by utilizing a variety of catalyst supports to constrain the particle.This therefore provides a big, suitable surface 18 .The heterogeneous catalyst needs a space so it won't dissolve into the solution matrix 19 .One of this catalyst is nano-CuO which used in this study.
An effective bioinformatic method known as PETRA/OSIRIS/MOLINSPIRATION (POM) analysis is used to evaluate the fundamental physical-chemical properties of molecules (represented as structures) and predict properties such as bioactivity, toxicity, and drug-likeness.This method's name is made up of the programs PETRA, OSIRIS, and the MOLINSPIRATION free online application.A software package called PETRA (Parameter Estimation for the Treatment of Reactivity Applications) consists of numerous empirical techniques for computing fundamental physicochemical parameters of organic compounds.The study team of Prof. J. Gasteiger has created all of the methodologies over the past 20 years, and they are all empirical in character.The heat of formation, bond dissociation energies, sigma charge distribution, π -charge distribution, inductive effect, resonance effect, delocalization energy, and polarizability effect may all be measured with this program 20 .
In the current reportnew composites of triazine and diazine derivatives were prepared and their activity as anti-Helicobacter pylori was studied and supported by computational studies (POM analysis and molecular docking) using MOLINSPIRATION, OSIRIS, ProTox-II and Pred-hERG software and using chemical computing groups of Molecular Operating Environment (MOE 2015) software.

Results and discussion
Characterization techniques of the copper oxide nanoparticles XRD analysis was used to determine the formation of CuO nanoparticles.The crystalline nature of the CuO-NPs and their matching phases were determined using XRD.XRD diffraction peaks at 2θ = 32.35, 35.62, 38.69,  48.72, 53.49, 58.33, 61.57, 66.31, 68.15, 73.12, and 76.15° were assigned in a good agreement to the monoclinic crystallite CuO (JCPDS-05-0661) planes ( 110), (002), ( 111), (−202), (020), (202) 21 as designated in Fig. 1.There were no further peaks attributable to suspected copper hydroxide [Cu(OH) 2 ] and/or Cu 2 O, demonstrating the purity of the high grade generated CuO-NPs with monoclinic crystal structure.As a result, it is possible to conclude that the XRD reveals a single phase monoclinic structure of CuO-NPs.The end result was comparable to statistics reported elsewhere 22 .
The HR-TEM image revealed detailed morphological information about the CuO-NPs (Fig. 2a and b).Particles were made up of a sheet-like building structure 23 .The nanoparticles coincided with one other which greatly aided the growth of the flower like nanostructure together with oval and spherical outlines 24 .Furthermore, a crystallographic experimental approach known as "Selected Area (Electron) Diffraction (SAED) pattern enclosed bright circular patches that corroborated the polycrystalline nature, implying that the CuO particles have various crystallographic directions, as seen in Fig. 2c 23 .
The structural analysis using SEM is presented in Fig. 3a, which displays the morphological progression of the CuO-NPs, which were discovered to be flakes or plate-like structures similar to the petals of a flower.The attained CuO floweret-nanostructures are talented nominee for possible use in catalysis 25 .The elemental chemical composition of the nanoparticles was investigated by EDX analysis (Fig. 3b).EDX analysis was used to determine the elemental chemical composition of the nanoparticles (Fig. 3b).It found that the chemical makeup was 49% copper and 51% oxygen in atomic percent.This finding confirmed that the CuO-NPs were pure 26 .This outcome was elucidated by microscopic evaluations and spectroscopy 25 .
The "Specific Surface Area (SSA)" is a distinctive substance that plays an important function in nanoparticles due to the large ratio of surface to volume with decreasing particle size.Adsorption, heterogeneous catalysis, and surface reactions all rely on it.The CuO-NPs' large surface area aided the reaction/interaction between CuO and the interacting media, which occurs often on the surface or at the interface and is influenced by the material's surface area.The surface area of the produced CuO-NPs was 58.4419 (m 2 /gm)was presented in Fig. 4, as sustained by Quantachrome TouchWinTM, model NOVA touch 4LX 27 .
To attain the finest kind of catalysts, sodium acetate (C 2 H 3 O 2 Na) and CuO-NPs production were correlated.It became apparent that the finest features were attained when the CuO-NPs was elaborated in the reaction.To amend the time, the model reaction was accomplished under stirring conditions.Organized results verified that 99% yield was reached after 4 min upon consuming of the CuO-NPs as a catalyst (Table 1).The tabulated results showed that CuO-NPs achieved best results in time and yield.
The IR spectrum of 2 demonstrated a broadband at 3444.87-3360 cm −1 , consistent to the OH in the carboxylic group, and the peaks at 1678, 1624and 1450 cm −1 were along with (2C=O for carboxylic group), (2C=O for ketones) and (2 N=N), respectively.For the 1 H-NMR it exhibited δ at 15.843 ppm for 2-OH which was replaceable by D 2 O. 13 C-NMR spectra exhibited the carbon of the carbonyl group (C=O) at 197.29 ppm and the carbon of acid at 168.21 ppm.MS (M/Z): molecular peak was elucidated at (396.56), while base-peak emerged at (207.28), reported in 29 .
The reaction of 2-((2,4-dioxopentan-3-yl)diazenyl)benzoic acid with different reagents deliberated as a preparatory point for the synthesis of new collections of compounds exhibiting numerous pharmaceutical activities, where heterocyclization of 2-((2,4-dioxopentan-3-yl)diazenyl)benzoic acid 2 by reacting with Dimadone & phenyl hydrazone in DMF in presence of TEA as basic medium provided  A promising mechanism for the reaction is presented in Fig. 6.The reaction of compound 2 with hydrazone, cyanoacetamide and cyano aceto hydrazide under reflux condition in the incidence of TEA resulted in the initiation of compounds (6, 7 & 8), respectively.For compound 6, IR demonstrated 3426 (OH), 3216(NH), 2927 (CH for aliphatic), 1717 (C=O of ketone), 1702 (C=O of acid), 1674 (C=O of amide), 1600 (C=N), respectively.As well, the 1 H-NMR spectra showed the appearance of the-3CH groups, 2NH and -NH 2 signals at δ 3.979, 5.387, 7.722, 7.340, 7.722, 7.378 ppm, respectively.Similarly, the structure of compounds 7 & 8 was offered by elemental analysis and spectral data observed in the experimental section Fig. 7.A possible mechanism for the reaction of compound 6 is revealed in Fig. 8.The reaction of compound 2 with 3-Oxo-N-phenyl butanamide, Acetyl Acetone and Ethyl acetoacetate, respectively, directed to the   formulation of compounds (9, 10 & 11), whose structure were provided by elemental analysis and spectral data as recognized in the experimental section, presented in Fig. 7.In Fig. 9, chemical 2 was refluxed with phenyl hydrazine and benzyl amine in DMF, which was grounded to produce compounds 12 & 13 respectively.Compound 12, structure was validated by its proper elemental analysis.Its IR spectra revealed broad functional group at 3057 cm −1 indicatives for the NH and the appearance of more aromatic ring in the 1 H-NMR spectra.While compound 13's structure was confirmed from its precise elemental analysis, the IR spectra, showed new functional group bands at 3078 cm -1 , broad one for NH and at 1600 cm −1 for the cyano group (C=N), and the appearance of more aromatic ring on 1 H-NMR spectra with vanishing of the azo group.When compound 2 reacted with benzilidine malononitrile and benzal aniline in refluxing DMF containing few drops of TEA, it provided desired compounds of 14 & 15, respectively as presented in Fig. 10.

Determination of inhibitions zones of samples
Results revealed that the inhibitions zones in millimeters of samples 2, 3, 4, 5, 7, 8, 10, 11, 12 and 13 on the Anti-H.Pylori were 22.33, 21.00, 19.67, 18.33, 23.67, 17.67, 22.00, 21.33, 23.33, and 25.00 mm, respectively as indicated in (Table 2).All compounds have bactericidal effect better than the reference McFarland standards (used to modify the turbidity of bacterial suspensions so as to the number of bacteria will be within a given range to homogenize microbial testing) which has an inhibition zone of 19.67 mm Fig. 11.DEMSO was used as a control negative as it was used for dissolving the samples and it had not any antibacterial activity.The data were analyzed using IBM SPSS Statistics (Version 27) 30 .
The anti-Helicobacter activity of newly synthesized compounds was assessed in relation to previous studies, Rüegg et al. 31 corroborated the discovery of a novel derivative, 3-farnesyl-2-hydroxy benzoic acid, in Piper multiplinervium leaves.This compound exhibited substantial anti-Helicobacter pylori activity at 37.5 µg/ml and demonstrated efficacy against various bacteria and fungi, including Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Mycobacterium smegmatis, Pseudomonas aeruginosa, and Candida albicans, with   www.nature.com/scientificreports/MICs ranging from 2.5 to 5 µg/ml.Its structure was elucidated using MS, 1 H, and 13 C NMR techniques.These findings support the potential use of benzoic acid derivatives in addressing stomach discomfort, aligning with observed anti-Helicobacter pylori activity.Compound 12 emerged as the safest option for anti-Helicobacter use, exhibiting a predicted LD 50 of 8000 mg/kg 32 and a toxicity class of six, as determined by the Protox II virtual lab for molecular toxicity analysis in a rat model.Additionally, Compound 12 displayed significant anti-Helicobacter activity, surpassing standard drugs with a zone of inhibition of 23.33 mm, MIC of 3.9 µg/ml, and MBC of 7.8 µg/ ml compared to 19.67 mm, 1.95 µg/ml, and 1.95 µg/ml for the standard drug, respectively.

Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC)
Table 3 elucidates that all the tested samples have bactericidal effect where MBC/MIC Index for all tested samples was ≤ 4. The index of MBC/MIC of samples 3, 8 & 12 was two folds that of other samples.

In silico studies
POM analysis POM analysis and similar methods are important tools for determining different physico-chemical characteristics and forecasting a molecule's biological activity, ADME parameters, and toxicity.Compounds 7, 12, and 13 were subjected to a modified POM analysis utilizing the MOLINSPIRATION, SWISSADME, and OSIRIS tools.The expected toxicity dangers for chemicals 7, 12, and 13 were calculated using the OSIRIS tool Fig. 12, and the results are shown in Fig. 12.According to normal clinical medications, these compounds produce less adverse effects.Additionally, it was shown that substances 7, 12, and 13 have some pharmacomodulation and may behave as antibiotics (DS = 0.53, 0.42, and 0.5, respectively) where toxicological for these compounds have some reproductive toxicity and have TPSA more than 140 Å 2 where (TPSA score ideal for drug-like molecules is less than 140 Å 2 ).Molinsipration calculation.A wide variety of computational biology programs are available through MOLIN-SPIRATION that help with the manipulation and processing of molecules.These tools include SMILES and Sdfile conversion, molecule normalization, tautomer generation, molecule fragmentation, calculation of various molecular properties required for QSAR, molecular modeling and drug design, high-quality molecule depiction, and molecular database instruments assisting substructure and likeness searches 22,33 .
Table 4 lists the anticipated pharmacokinetic/Molinspiration parameters for the chemicals produced 7, 12, and 13.With the use of Molinspiration online screening, almost all of the compounds produced had potential biological activity, as demonstrated by the docking parameters in Table 5, which highlight the drug-like properties against kinase inhibitors, protease, and enzyme inhibitors.The Calculated distribution of activity scores (version 2022.08) are compared to scores for GPCR ligands, kinase inhibitors, ion channel modulators, nuclear   Pred-hERG.Biologically varied protein targets are frequently bound by chemically related substances, although protein structures may not always recognize the same ligands.By interpolating the output prediction equalized by the compound similarity criteria, pharmacological and off-target connections between proteins and a ligand set assist increase the machine learning confidence.This pipeline contributes to lowering the falsenegative error and improving forecasts of off-target medication effects.One of the key ideas in cheminformatics is chemical similarity.The 2D Tanimoto technique utilized here is one that is frequently used to determine these similarity algorithm metrics.The final Tanimoto coefficient is fingerprint-based, encoding each molecule to a fingerprint "bit" location (MACCS), with each bit recording whether a molecule fragment is present ("1") or not ("0") in the sample.The potency results are represented in Table 6b, While Fig. 14 shows the Probability Map of compounds 7, 12, 13.

Molecular docking study
Molecular docking analysis was directed to study the contact of the natural compounds with several molecular targets of anti-inflammatory activity.Molecular docking analysis for the generated database was used to investigate the hypothesized mechanism of achievement for the newly developed and produced drug for bactericidal activity against H. pylori compared to a standard reference (4R,6R,7S)-2-(2-cyclopropylethyl)-4,6,7-trihydroxy-4,5,6,7-tetrahydro-1-benzothiophene-4-carboxylic acid [JPS] (PDB code: 2XDA) 34 .Such analysis was carried out to obtain further insight into the binding modes of the synthesized compounds into the protein-binding site of type II dehydroquinase enzyme.
To confirm the current docking investigation at the active site, the co-crystallized ligand JPS was re-docked utilizing a similar collection of parameters.The best-docked pose's root mean square deviation (RMSD) was

Preparation of CuO-NPs
As an initiator, a suitable amount of copper nitrate [Cu(NO 3 ) 2 ] was liquefied in distilled water as a solvent.A reducing agent of 0.1 M sodium hydroxide (NaOH) was gradually added until the pH reached 12 at 50 °C while magnetic stirring (400 rpm) for one hour.The acquired blue precipitate was permitted to stand for 24 h till the color changed from blue to black.The resulting black precipitate was rinsed many times with deionized water until the pH reached 7. As a result, the cleaned precipitate was dehydrated for 6 h in an electric furnace at 100 °C.The produced CuO-NPs were investigated using (XRD), SEM/EDX, and the crystallinity of the nanoparticle was determined using (HR-TEM) and Specific Surface Area (SSA).

Characterization techniques
CuO-NPs were subjected to XRD, HR-TEM and SEM/EDX to recognize the particle characteristics, which closely coordinated with JCPDC standard.CuO phase identification and phase clarity evaluation were performed using XRD, enhancement of crystalline phases and evaluation of the produced sample's nano-size.XRD was performed using [Bruker D8 advance diffractometer, Germany] with monochromatized Cu K α (λ = 1.542Å) radiation in the scope of scattering angle (2θ) in the range of 5-80°.The nature and crystallinity of the nanoparticle were verified by high resolution transmission electron microscope [(HR-TEM), Joel model JEM-2100, Japan].The aqueous dissipation of the particulate was drop-casted onto a copper grid that had been coated with carbon and allowed to air dry at ambient temperature before being carefully considered.The micron-sized structure and appearance of the CuO was presented using scanning electron microscope equipped with energy dispersive x-ray microanalysis (SEM/EDX, model FEJ Quanta 250 Fei, Netherlands) operating at voltage 15 kV.The samples' surface was coated by gold by a [S150A sputter coater, Edwards, England] under 50 mA current, 0.1 Torr and vacuum 1.2 kV voltage.The gold concealing was to improve the samples' scanning.In the nanoparticles, the specific surface area (SSA) played an important character, because of the high ratio of surface to volume accompanied by a decrease in the dimension of particle.It has an accurate implication in case of the reactions on surfaces, adsorption, and heterogeneous catalysis.The surface area of the CuO-NPs permitted the overall size of the material significantly influenced the chemical reaction between the CuO and the substrate, which typically takes place at the interface or on the surface.In present research, surface area of CuO-NPs was 58.4419 (m 2 /g), which was restrained via Quantachrome Touch WinTM, model NOVA touch 4LX, using nitrogen as adopant 35 .

2-(
, 0.005 mol) was dissolving in (9 mL) dimethylformamide then was mixed with a solution of cyanoaceto hydrazide (convenient intermediate for the production of diversity of heterocyclic compounds) (0.49 g, 0.005 mol) in (9 mL) dimethyl formamide in the occurrence of few drops of triethylamine (1 ml).The reaction components were heated under reflux for 17 h, the reaction was followed by TLC system using mixture of methylene chloride as eluent.The dissociated crystal was filtered out and crystallized by a combination of DMF and ethanol by ratio (2:1) after being refrigerated for a night and poured progressively over broken ice by ratio (2:1) to give off compounds 8 as black powder: 78%, m.p.:237 °C.IR (ν, cm −1 ): 3449 (OH), 3070(broad band of NH 2 and NH), 1717 (C=O of ketone), 1702 (C = O of acid), 1676 (C=O of amide).0012 mol) and 3-oxo-N-phenyl butanamide (0.212 g, 0.0012 mol) were dissolved in (20 mL) dimethylformamide in presence of few drops of triethylamine (1 mL), The TLC system used a mixture of methylene chloride as the eluent for observing the reaction.After cooling and being poured over ice that had been crushed, the fluid was refluxed for 15 h.To create compounds 9, the produced precipitate was collected and crystallized with a solution of DMF and ethanol in a 2:1 ratio, yellow powder of compound
The precipitate was dried and recrystallized using a 2:1 combination of DMF and ethanol to yield compound 13 as a brown powder 76% with a melting point of 228°C.IR (ν, cm

2-((1-acetyl-2,2-dicyano-5-oxo-3-phenylcyclopent-3-en-1-yl)diazenyl)benzoic acid (14):
In the existence of a few drops of triethylamine, a solution of compound 2 (0.50 g, 0.0012 mol) in (11 mL) dimethylformamide was mixed with a solution of benzilidine malononitrile (0.185 g, 0.0012 mol) in (11 mL) dimethylformamide.The resulting mixture was refluxed for 17 h, the interaction was monitored by TLC process using methylene chloride as eluent, then allowing it to cool for a night and poured gradual over ice cubes, the isolated crystal was filtered off and recrystallized by mixture of DMF and ethanol by ratio (2:1) to give off compound  36 .This turbidity yields a suspension that relates to approximately 1.0 × 10 8 CFU/mL of H. pylori.Verification of anti-H.Pylori action: The in vitro anti-H.pylori activities were established through well agar diffusion method 37 .Temporarily, 100 μL of H. pylori suspension (1.0 × 10 8 colony forming units (CFUs)/mL) was laid out onto Mueller Hinton agar plates (BBL) holding 10% sheep blood.At that time, a ditch of 6-8 mm diameter is perforated using a sterile cork borer, and a 100 μL volume of the antimicrobial agent otherwise solution extract at chosen concentration is presented into the well.The negative control is dimethyl sulfoxide (DMSO), whereas the positive ones are clarithromycin (CLR, 0.05 mg/mL), antibiotics amoxicillin (AMX, 0.05 mg/ml) and metronidazole (MTZ, 0.8 mg/mL).Afterward, maturation of 72 h at 37 °C under a microaerophilic condition by means of humidity, the inhibition zone diameter (IZD) was set on.
Minimal inhibitory concentration (MIC).The micro-dilution broth method, using Mueller-Hinton broth supplemented with lysed horse blood, allowing for the determination of the minimal inhibitory concentration (MIC) of the tested samples.Serial two-fold dilutions were made in order to obtain final concentrations of the tested samples, which ranged from 0.98 to 1000 μg/mL.The sterile 96-well polystyrene microtitrate plates were prepared by dispensing 200 μL of appropriate dilution of the tested samples in broth medium per well.The inocula were prepared with fresh microbial cultures in sterile 0.85% NaCl to match the turbidity of 1.0 McFarland standard, and 2 µL were added to the wells to obtain a final density of 3.0 × 10 6 CFU (colony forming units)/mL.After incubation at 35 °C for 72 h under microaerophilic conditions (15% CO 2 ), the MICs were assessed visually as the lowest concentration of the tested samples showing complete growth inhibition of the reference strain.A positive control (containing inoculum without the tested samples) and a negative control (containing the tested samples without inoculum) were included on each microplate ("Supplementary Information").
Minimal bactericidal concentration (MBC).MBC was determined by sub-culturing 100 mL of the microbial culture from each well that showed thorough growth inhibition, from the last positive and from the growth control, onto the plates of Mueller-Hinton agar supplemented with 5% horse blood.The plates were incubated at 35 °C for 72 h under microaerophilic conditions, and the MBC was defined as the Lowes concentration of the tested samples without growth of microorganisms.To govern the bactericidal or bacteriostatic effect of the evaluated samples, the ratio of MBC/MIC was considered [38][39][40] .

Insilco studies
The tools used for POM analysis, especially OSIRIS, MOLINSPIRATION 39 , ProTox-II 40 and Pred-hERG [41][42][43][44] , operate using the same basic concept.The molecular structure is initially determined either by sketching it or by entering its SMILES (simplified molecular input line entry system) code.The specific parameters are then determined using the fragment system [45][46][47][48] , then the software of the molecular operating environment was used to perform the molecular modeling for the highest active compounds  .

Conclusion
Aneco-friendly and cost-effective, engagement has been evolved for the fabrication of 2,2′-((2,4-dioxopentane-3,3-diyl) bis(diazene-2,1-diyl) dibenzoic acid 2 in the presence of CuO-NPs to start a fast and ecologically friendly procedure.Cyclization of 2 cause the creation of new families of triazine and diazine derivatives.Moreover, the recently combined chemicals were verified as anti-H.Pylori activity.All tested samples have a bactericidal effect showing MBC/MIC Index for all tested samples ≤ 2. Conclusively, authors considered the molecular docking tentative study, for the synthesized compounds 7, 12 and 13.These compounds were proved to betalented candidates for supplementary studies to be used as an effective, efficient and safe anti-H.pylori medication.This study paves the way for this nanoparticle material as an as anti-H.Pylori therapy.

Figure 2 .
Figure 2. HR-TEM images of CuO-NPs (a & b) Micrograph image illustrate particles with sheet-like structures, (c) Diffraction pattern elucidate the crystallinity of sample.

Figure 8 .
Figure 8.The plausible mechanism for the synthesis of compound 6.

Figure 9 .
Figure 9. Reaction of compound 2 with phenyl hydrazine and benzyl amine reagents.

Figure 13 .
Figure 13.The oral toxicity prediction results &the toxicity radar chart is intended to quickly illustrate the confidence of positive toxicity results compared to the average of its class for compounds 7, 12 and13.

Figure 15 .
Figure 15.2D receptor interactions and 3D receptor interactions of the ligand JPS as a reference for bactericidal activity against H. Pylori.

Figure 16 .
Figure 16.2D receptor interactions of the promising synthetized compounds against H. Pylori.

Figure 17 .
Figure 17.3D receptor interactions of the promising synthetized compounds against H. Pylori.

Table 1 .
Optimization of the reaction conditions.

Table 3 .
Determination of samples concentration (MIC) and (MBC).NB: If the MBC/MIC index of the samples ≤ 4 suggested their bactericidal activity against H. pylori, while the MBC/MIC index of the samples > 4 demonstrated their bacteriostatic activity.

Table 4 .
Physicochemical properties of the synthesized compounds.receptorligands,proteaseinhibitors,andother enzyme targets.These scores contain scores for over 100,000 common druglike compounds.Effective differentiation between active and inactive molecules is made possible by the score.ProTox-II and Pred-hERGProtox II virtual lab for the analysis of little molecule toxicity.Identifying chemical toxicity is a crucial step in the creation of new pharmaceuticals.According to the ProTox-II, the oral LD50 values for the three chemicals in a rat model range from 159 to 2480 mg/kg, with quercetin having the lowest value and (1 s, 4 s)-Eucalyptol having the highest.Figure13displays the comparison of chemicals 7, 12, and 13 to those in the dataset where Predicted toxicity class for compound 12 where 6 which have high Predicted LD50 (mg/kg).

Table 7 .
The binding scores, RMSD values, distance, receptor interactions of the most three promising compounds (7, 12 and 13) compared to the reference ligand [JPS] as a reference for bactericidal activity against H. pylori.